The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use 5.1µl for 106 cells.
ab106163 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
Use a concentration of 1 µg/ml.
Microtubules are involved in a wide variety of cellular activities ranging from mitosis and transport events to cell movement and the maintenance of cell shape. Tubulin itself is a globular protein which consists of two polypeptides (alpha and beta tubulin). Alpha and beta tubulin dimers are assembled to 13 protofilaments that form a microtubule of 22 nm diameter. Tyrosine ligase adds a C-terminal tyrosin to monomeric alpha tubulin. Assembled microtubules can again be detyrosinated by a cytoskeleton associated carboxypeptidase. Detyrosinated alpha tubulin is referred to as Glu-tubulin. Another post-translational modification of detyrosinated alpha tubulin is C-terminal polyglutamylation which is characteristic for microtubules in neuronal cells and the mitotic spindle.
Alpha tubulin is not suitable as a loading control in adipose tissue as expression of tubulin in adipose tissue is very low ( Spiegelman and Farmer, Cell, 1982, 29(1): 53-60, "in cells undergoing adipose differentiation actin synthesis decreases by 90%").
ICC/IF image of ab64503 stained human HeLa cells. The cells were methanol fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab64503, 1µg/ml, FITC conjugated (green)) for 1h at room temperature. 1% BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
Immunohistochemistry (Frozen sections) - Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (FITC) (ab64503)This image is a courtesy of Abreview submitted by Anonymous
ab64503 staining alpha Tubulin in Xenopus laevis stage 36, transverse cryosection tissue section by Immunohistochemistry (Frozen sections). Tissue samples were fixed with MEMFa and blocked with 2% BSA/Serum for 1 hour at 230C. The sample was incubated with primary antibody (1/100) for 1 hour at 230C. No secondary antibody was used. FITC-tubulin shown in green highlights neurons in neural tube and ciliated epidermal cells. DAPI shows staining in grey. Laminin (Abcam`s ab11575) is in blue (donkey anti-rabbit Cy5 secondary). E-cadherin is in red (donkey anti-mouse Cy3 secondary).
Overlay histogram showing HeLa cells stained with ab64503 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab64503, 0.5µg/1x106 cells) for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 (1µg/1x106 cells ). Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Tween used under the same conditions.